PMID: 9531509Jun 11, 1998Paper

Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C

The Biochemical Journal
P BorowskiR Laufs

Abstract

The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-...Continue Reading

Citations

Apr 29, 2015·Cell Biochemistry and Function·Cristiana Carelli-AlinoviFrancesco Misiti
Aug 1, 2012·PloS One·Mei-Ying ChangWan-Wan Lin
Dec 7, 2013·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Betina S FoghJohn R Couchman
May 31, 2001·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·T HitomiH Yamamura
Jul 15, 2009·The Journal of Immunology : Official Journal of the American Association of Immunologists·Oana Popa-NitaPaul H Naccache

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